| Cyclodextrin glycosyltransferase, abbreviation is CGTase, it can turn oligosaccharides to match-mismatch sugar by glycosyl reaction, besides, it can modifiy sweet chrysanthemum sugar, through the ring reaction it can convert starch to cyclodextrins, due to the unique characteristic and special applications of these materials, CGTase has great applications in food areas and so on.In our eraly study, we have made some progress in expression of Paenibacillus maceransα-CGTase in recombinant Escherichia coli. In this study, P. maceransα-CGTase was expressed in Pichia pastoris and Bacillus subtilis, which were widely used in industry, a series of feasible fermentation optimization were carried out to achieve higher productivity ofα-CGTase. The main results are listed as follows:(1) Theα-cgt gene isolated from P. macerans was cloned in the P. pastoris vector pPIC9K. Thus, the expression plasmid pPCGT was constructed and transformed into the host P. pastoris KM71, a recombinant strain P. pastoris KM71/pPCGT was developed to produceα-CGTase. However, the activity ofα-CGTase in the culture medium was only 0.2 U/ml after cultivation in shaking flask for 96 h. There were no visible bands in SDS-PAGE. The codon preference and glycosylation were analysed. The low frequency codon usage of P. pastoris in the cgt gene is 8%, and there are nine glycosylation sites in the cgt gene, which indicated poor codon usage and glycosylation may have effect on the expression of CGTase in P. pastoris.(2) Theα-cgt gene was cloned in the B. subtilis vector pMA5. Thus, the expression plasmid pMCGT was constructed and transformed into the host B. subtilis WB600. The extracellular activity of the recombinantα-CGTase in B. subtilis WB600/pMCGT was 1.9 U/mL for 36 h, and there was no CGTase acitivity in the control experiment of WB600/pMA5. Besides, there was no cellular insoluble fraction by SDS-PAGE. The culture conditions for recombinat strain were optimized in shaking flask and optimal conditions were:an initial medium of TB medium, temperature at 37℃, an initial pH of 7 and an inoculum size of 4%. Theα-CGTase activity achieved 4.3 U/mL at this condition.(3) Theα-cgt gene was then cloned into the B. subtilis induciton vector pGJ103 and it was transformed into the host B. subtilis WB600, then a recombinant strain B. subtilis WB600/pGCGT was developed. After induction with 1% maltose, theα-CGTase activity was 5.1 U/mL. SDS-PAGE also showed that the objection proteins were included in supernant. We investigated the seed growth, the logarithmic growth phase was from 4 to 12 h, and the seed age was 8 h. Besides, we investigated the stability, it was still over 92% after five subcultures.(4) The culture medium for B. subtilis/pGCGT in shaking flask was investigated. According to the single-factor experiments, malose concentration was 15 g/L, corn starch and yeast extract were identified as optimal carbon source and nitrogen source, respectively. The expression level of CGTase of 17.6 U/mL could be obtained when the mediume consisted of maltose 15.5 g/L, corn starch 13 g/L, yeast extract 20 g/L, peptone 4 g/L, NaNO3 2 g/L, K2HPO412.4 g/L, KH2PO42.3 g/L。(5) The fermentation of B. subtilis/pGCGT in a 5 L stirred fermentor was studied. Through the batch experiments we found that when the DO concentration was 20%, it was benifitial for better cell growth and a-CGTase production. On the basis of batch fermentation, when fed with 20 g/L sucrose, the maximum DCW achieved 15.5 g/L, the final a-CGTase activity was 30 U/mL after 35 h cultivation.
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